WO1999013320A1

WO1999013320A1 - Optical sensor and optical method for characterizing a chemical or biological substance

Info

Publication number: WO1999013320A1
Application number: PCT/CH1998/000389
Authority: WO
Inventors: Kurt Tiefenthaler
Original assignee: Artificial Sensing Instruments Asi Ag
Priority date: 1997-09-10
Filing date: 1998-09-10
Publication date: 1999-03-18
Also published as: US6787110B2; US7867448B2; EP1443321A2; US6958131B2; US20050201657A1; US6455004B1; DE59811600D1; US20030007896A1; EP1443320A2; EP1012580A1; EP1443320A3; EP1443321A3; EP1012580B1; US20040247486A1

Abstract

The invention relates to an optical sensor comprising an optical waveguide (1) with a substrate (104), wave guiding material (105), a cover medium (106) and a wave guide mesh effect (101-103). Light can be transmitted by means of a light source (2) from the substrate side and/or from the cover medium side to the waveguide mesh effect (101-103). At least two different light portions (7-10) radiated from the waveguide (1) can be detected by detection means (11). In order to conduct a measurement, the waveguide (1) can be fixed in a stationary manner with regard to the light source (2) and the detection means (11). The waveguide mesh effect (101-103) is comprised of one or more waveguide mesh effect units (101-103) which can be provided with (bio)chemical layers. The sensor permits generating of absolute measuring signals.

Description

OPTICAL SENSOR AND OPTICAL METHOD FOR

CHARACTERIZATION OF A CHEMICAL AND / OR

BIOCHEMICAL SUBSTANCE

The invention relates to an optical sensor and an optical method for characterizing a chemical and / or biochemical substance.

Waveguide grating structures with and without a chemo-sensitive layer are described in the literature (see, for example, EP 0 226 604 B1, EP 0 482 377 A2, PCT WO 95/03538, SPIE Vol. 1141, 192-200, PCT WO 97/09594, advances in biosensors 2 (1992), 261-289, USP 5'479'260, SPIE Vol. 2836, 221-234).

EP 0 226 604 B1 and EP 0 482 377 AI show how the effective refractive index (or the coupling angle) of a chemosensitive grating coupler can be measured as a sensor signal. The sensor signal "effective refractive index" or "coupling angle" is a variable that has a strong temperature dependence.

Front coupling of light into a waveguide (see SPIE Vol. 1141, 192-200) is impractical because high positioning accuracy is required. In addition, the end face of the waveguide must have optical quality. PCT WO 95/03538 shows how the absolute coupling angle of a mode is measured. However, this size shows a strong temperature dependence without referencing. PCT WO 97/09594 presents chirped waveguide gratings, which, however, also show a dependence on the temperature. In Advances in Biosensors 2 (1992), 261-289 it is shown how the "pore diffusion" disturbance can be referenced away with the three-layer waveguide model. The refractive index of the waveguiding film shows drift, while the layer thickness of the waveguiding film (= sensor signal) remains stable. The arrangement is constructed with movable mechanics, which does not allow quick measurements. In addition, the sensor signal or the light emerging from the waveguide grating structure is detected on the end face. Frontal detection is unsuitable for a two-dimensional arrangement of waveguide grating structure units. Furthermore, the effective refractive indices N (TE) and N (TM) for the two polarizations TE and TM are not recorded simultaneously, since a mechanical angle scan is carried out to record the angularly separated resonance coupling curves.

USP 5'479'260 describes a bidif fractional or multidifractive grating coupler, the sensor signal being obtained by interferometry of two outcoupled beams of the same or different polarization (using a polarizer). Interferometric measurements are complicated because the intensities of the two beams have to be coordinated. In addition, temperature fluctuations are only partially compensated for by the interferometric signal generated by different polarizations (using a polarizer).

An arrangement for a waveguide grating structure in connection with fluorescence or luminescence measurements is described in SPIE Vol. 2836, 221-234. However, this arrangement is not suitable for a (possibly simultaneous) (absolute) temperature-compensated measurement based on direct verification. In addition, the waveguide grating structure is mounted on a turntable.

Applied Optics 20 (1981), 2280-2283 reports on a temperature-independent optical waveguide, the substrate being made of silicon. Silicon is absorbent in the visible spectral range. With chemosensitive waveguide gratings However, structures are preferably coupled in from the substrate side. Applied Optics 20 (1981), 2280-2283 also deals with grid couplers that are not temperature-independent.

The object of the present invention is to create a (bio) chemo-sensitive optical sensor and to provide an optical method for characterizing a (bio) chemical substance which does not have the above disadvantages. In particular, the invention is intended to:

(1) sensor signals which have a low temperature dependency and / or a slight dependence on the diffusion of the sample liquid into the micropores of a waveguiding film can be generated,

(2) both the measurement of (absolute) sensor signals with regard to direct detection (absolute coupling angle α (TE) and α (TM) for the TE and TM wave, effective refractive indices N (TE) and N (TM) for the TE or TM wave, layer thickness t _{F of} the wave-guiding film etc.) and also the measurement of (absolute) sensor signals with respect to a marking detection (referenced fluorescence, luminescence, phosphorescence signals etc.) are possible and

(3) Sensor signals remain stable with respect to a slight tilting and / or shifting of the waveguide grating structure, since (local and angular) differences of sensor signals or referenced sensor signals are measured.

The object is achieved by the invention as defined in the independent patent claims.

The optical sensor according to the invention contains at least one optical waveguide with a substrate, waveguiding material, a cover medium and at least one waveguide grating structure, at least one light source, by means of which light can be transmitted from the substrate side and / or from the cover medium side to at least part of the waveguide grating structure , and means for the detection of at least two different light components, of which light emitted into the substrate and / or the cover medium can be detected with at least one detection means, the waveguide being immovably fixable with respect to the at least one light source and the detection means for carrying out a measurement.

In the optical method according to the invention for characterizing a chemical and / or biochemical substance in a sample by means of an optical waveguide containing at least one waveguide grating structure, the sample is brought into contact with the waveguide in at least one contact zone, at least one in the waveguide in the region of the at least one contact zone Light wave excited via the waveguide grating structure, which interacts at least one light wave with the sample, detects light in at least two different proportions, at least a portion of which comes from the area of the contact zone, and generates at least one absolute measurement signal by evaluating the detected light.

The waveguide grating structure consists of one or more waveguide grating structure units which are arranged one-dimensionally or two-dimensionally (e.g. matrix-shaped or circular).

A possible xy shift (or only x shift) of the read head (the read heads) from one waveguide grating structure unit to another or a possible xy shift (or only x shift) of the waveguide grating structure can certainly be used.

A waveguide grating structure unit consists of at least two sensing pads (sensor platforms, sensor paths), which differ from one another by at least one of the following features:

(a) The light waves guided in the 'sensing pads' differ in polarization (TE wave or TM wave), whereby the sensor signal generated does not come about through interferometric measurement (b) those in the 'sensing pads' Light waves differ in the number of modes. (c) The two chemo-sensitive layers assigned to the 'sensing pads' show different specificity (ligand 1 binds (inside or on the surface) selectively to the chemosensitive layer 1; ligand 2 which covers the 'sensing pad 1' (inside or on the surface) selectively to the chemosensitive layer 2 covering the 'sensing pad 2')

(d) The chemosensitive layer assigned to the first 'sensing pad' shows specificity for a ligand (with or without 'non-specific binding'), while the (chemosensitive) layer assigned to the second 'sensing pad' shows no specificity (with or without ' non-specific binding ') (example: dextran layer to which no recognition molecule (eg an antibody) is bound).

(e) The light waves in the 'sensing pads' differ in wavelength.

A 'sensing pad' in which guided light waves of different polarization (TE wave or TM wave) are excited counts as two 'sensing pads' (difference in polarization!), Provided that the sensor signal generated is not by interferometric measurement comes about.

A 'sensing pad' in which guided light waves of different mode numbers are excited counts as two 'sensing pads' (difference in mode number!).

A 'sensing pad' in which guided light waves of different wavelengths are excited counts as two 'sensing pads' (difference in wavelength!).

The first and second 'sensing pad' can also be understood as a signal and reference path. The two 'sensing pads' can (but do not have to) have the same structure.

The (bio) chemo-sensitive layer contacts the waveguiding film in a contact zone. For direct detection, this contact zone normally contains at least one grating (for interferometric measurements with the same polarization, for a direct detection the (bio) chemo-sensitive layer is only between two grids (see also EP 0 226 604)) (for interferometric measurements with two different polarizations (using a polarizer) the (bio) chemo-sensitive layer can also be placed on a multidiffractive ( bidiffractive) grid (see USP 5'479'260)).

In principle, the size S (signal path) - cS (reference path) can serve as a possible referenced sensor signal, with S (signal path) and S (reference path) the sensor signals in the first 'sensing pad' (signal path) and in the second ' sensing pad 'and c are a calibration factor. With the same polarization, c = 1 is reasonably. For different polarizations, the different sensitivities of the two polarizations can be taken into account with c. With different wavelengths or mode numbers, the different sensitivities of the wavelengths or mode numbers can be taken into account with c. The signal path and reference path are advantageously as close as possible.

The referenced sensor signal with the same polarization also has the advantage that interference δ such as those caused by temperature fluctuations, light wavelength fluctuations, undesired diffusion of molecules into the waveguide or into the chemosensitive layer, non-specific bonds, fluctuation in the concentration in the undetectable molecules etc. or combinations thereof can be referenced, i.e. the referenced sensor signal is independent of δ, since S (signal path) + δ - (S (reference path) + δ) = S (signal path) -S (reference path).

Monomode waveguides are preferably used which carry only the fundamental TE mode or only the fundamental TE mode and the fundamental TM mode. The wave-guiding film should preferably consist of a highly refractive material, which guarantees the production of high sensitivities. The waveguiding film can be coated with a chemo-sensitive layer (eg an antibody layer (eg suitable for the detection of a corresponding antigen), a dex- tran layer with a recognition molecule (eg antibodies), receptors, DNA sections, a silicone layer for the detection of hydrocarbons, etc.). However, the waveguiding film itself can also be a chemo-sensitive layer. Strip waveguides can also be used.

A 'sensing pad' comprises at least one grating, but can also comprise at least one (possibly more modulated) coupling grating and at least one coupling grating. The grating periods of the two sensing pads can (but do not have to) be different. The grating periods of the coupling-in grating and the coupling-out grating can be different (in most cases they are different).

The coupling grating and the coupling grating can be unidiffractive or multidirectional grating structures (bidioactive grating, grating with changing grating period and / or with changing grating diffraction vector, etc.).

A preferred 'sensing pad' arrangement consists of three gratings, the central grating representing the coupling-in grating and the two outer gratings representing two coupling-out gratings. With a strongly modulated coupling grating, for example, it is possible to excite modes in the forward and backward directions with a single (or with two) incident (possibly slightly focused) light beam (s). A slight shift of the incident light beam in the direction of mode propagation or a slight tilting of the waveguide grating structure with respect to the incident light beam (in the plane of incidence) change the intensity of the modes (running in the forward and / or backward direction) as a result of changed coupling geometry, but not the (the) decoupling angle or the difference of the coupling angle (= double absolute coupling angle), which represent possible sensor signals.

FIG. 1 shows the above preferred 'sensing pad' arrangement both for the TE mode and for the TM mode, the two 'sensing pads' lying next to one another. FIG. 2 shows further waveguide grating structure units.

FIG. 3 shows an optical sensor according to the invention.

Another preferred 'sensing pad' arrangement also consists of three gratings, the two outer gratings (possibly more modulated) coupling gratings and the middle grid forming the coupling gratings.

In the detection of (bio) molecular interactions with the arrangement according to FIG. 1 (or equivalent arrangements), the waveguide structure unit is coated with a (bio) chemo-sensitive layer, to which a specific binding partner then binds in the experiment, which leads to a change in the coupling angle α (TE) = (α (TE +) - α (TE-)) 2 and α (TM) = (α (TM +) - α (TM -)) / 2 (notation: e.g. α (TE +): decoupling angle of the in -b -Direction of running TE modes) or to a change in the effective refractive indices N (TE) and N (TM) and the derivable integrated optical parameters - such as the layer thickness tF of the waveguiding film in the three-layer waveguide model (see below). Different waveguide grating structure units can be covered with different chemo-sensitive layers.

The decoupling angles of the two decoupling gratings (or the decoupling grating) of the preferred 'sensing pad' arrangement allow an absolute determination of the decoupling angle (or the effective refractive index), although the decoupling of the two light beams does not take place at the same location.

In the case of two adjacent sensing pads for the TE and TM modes according to the preferred sensing pad arrangement (see FIG. 1), the coupling is even carried out at four different locations.

The sensing pad for the TE mode allows the effective refractive index N (TE) of the TE mode to be determined. The 'sensing pad' for the TM mode allows the determination the effective refractive index N (TM) of the TM mode. With simultaneous illumination of the two 'sensing pads' (e.g. with a single light field with 45 ° linearly polarized light or circular (or elliptically) polarized light) the determination of the decoupling angle (the effective refractive index) for the TE and TM modes can done early.

FIG. 1 shows an advantageous embodiment of a waveguide grating structure unit. The 'sensing pad' for the TE mode consists of the coupling grating Gi (TE) and the two coupling grids Go + (TE) and G _o - (TE) located on the left and right of the coupling grid. The 'sensing pad' for the TM mode consists of the coupling grid Gi (TM) and the two coupling grids Go + (TM) and Go- (TM) located to the left and right of the coupling grid and is located next to the 'sensing pad' for the TE- Fashion. In order to keep the influence of disturbances (eg temperature fluctuations) low, the two 'sensing pads' should be as close as possible to each other. The grid lines are aligned parallel to the y-axis.

It is advantageous if the coupling grids G _o + (TE), G _o - (TE), G _o + (TM), G _o - (TM) are completely illuminated by the guided light waves, which ensures that the respective coupling grating the sensor surface corresponds to the surface of the decoupling grid. This is achieved in that (a) the width of the coupling grating in the y direction is chosen to be smaller / equal than the width of the coupling grating in the y direction and (b) the illumination spot on the coupling grating has a y width that is greater / is equal to the y-width of the coupling-out grating, and the illumination spot includes the entire y-width of a coupling-out grating.

An advantageous embodiment is that in which the two coupling gratings Gi (TE) and Gi (TM) are simultaneously illuminated with one (or two) (wedge-shaped) light field (s) linearly polarized at 45 °. This enables the TE mode and the TM mode (in the forward and backward direction) to be excited at the same time and all (in the present case: four) decoupling angles can also be will measure. Gi (TE) and Gi (TM) preferably have different grating periods.

However, it is also possible for a sensing pad to perform the function of a sensing pad for the TE mode and the function of a sensing pad for the TM mode. The second (adjacent) 'sensing pad' is then either not available or is used as a control or reference (this reference 'sensing pad' can, for example, with a second chemo-sensitive layer or a non-specific layer, ie a layer that has no specificity (with or without 'non-specific binding'). If the coupling grating of a 'sensing pad' is illuminated with TE light as well as with TM light (for example with 45 ° linearly polarized light) under the corresponding coupling angles, then in one sensing pad both the TE mode and the TM mode (possibly in both the forward and reverse directions).

It is also possible to excite the TE mode only in the forward direction and the TM mode only in the reverse direction (or vice versa) or the TE mode and the TM mode only in the forward direction (or vice versa). The corresponding two decoupling angles or their difference are then measured.

1 or 2-dimensional (digital or analog) position-sensitive detectors (PSD), photodiode arrays, CCD line or area cameras (s) etc. are advantageously used as detectors. The outcoupled light beams are advantageously focused with a lens, the focus not necessarily having to be exactly on the detector surface. The decoupling gratings can also be focusing gratings. This has the advantage that the lens effect is already integrated in the decoupling grating.

It is also possible to choose the direction of the grating lines of the coupling-out grating such that the grating lines are not perpendicular to the direction of propagation of the mode. Advantageously, the tilting of the grating lines (or the grating tion vectors) of the decoupling grating of two adjacent 'sensing pads' reversed sign. This enables a better separation of the outcoupled light waves.

The coupling grating can also be selected such that the grating period in the x direction or y direction or x and y direction does not remain constant or is continuous. This relaxes the requirements for the accuracy of the setting of the angle of incidence.

The coupling grids shown in FIG. 1 advantageously have a strong modulation and are distinguished by a short width in the x direction. As a result, the requirements for the accuracy of the setting of the angle of incidence are relaxed, since light can now be coupled in from a larger angle segment.

Figures 2a) and 2b) show two more

of waveguide grating structures, the extension of the coupling grating in the y direction being smaller than that of the coupling grating. FIG. 2b) shows a uniform decoupling grating Go- for the TE and TM waves running in the (-x) direction and a uniform decoupling grating Go + for the TE and TM waves running in the (+ χ) direction.

FIG. 3 shows an embodiment of a sensor according to the invention. The sensor contains a waveguide 1 with a substrate 104, a waveguiding material 105 and a cover medium 106 and with a waveguide grating structure according to FIG. 1. A light source 2 generates, for example, 45 ° linearly polarized light; For this purpose, a linearly polarized laser with a polarization plane tilted by 45 ° to the drawing plane can be used. Two incident light beams 5, 6 are generated via a beam splitter 3 or a mirror 4. The light rays 5, 6 fall through the substrate 104 onto the two coupling grids 101 (only one of which is visible in the side view of FIG. 3) of the two sensing pads (cf. FIG. 1) and produce in the first, sensing pad 'is a TE wave in the forward and reverse directions and in the second, sensing pad' is a TM wave in the forward and reverse directions. The Light waves are coupled out of the waveguide grating structure via four decoupling gratings 102, 103 (only two of which are visible in the side view of FIG. 3) and emitted into the substrate 104. After passing through the substrate 104, they spread out as light fields 7-10 and fall onto a two-dimensional CCD detector 11. A lens system (not shown) can optionally be located between the waveguide 1 and the detector 11.

If work is carried out in the two 'sensing pads' with different polarization and a chemosensitive layer assigned to the waveguide grating structure unit, then the sensor signal S = t _F (= layer thickness of the waveguiding film in the three-layer waveguide model) can be generated (see also below) . If a second waveguide grating structure unit, which also permits the generation of the sensor signal S = tF, is now placed next to the first waveguide grating structure unit (now to be considered as a signal path in its entirety) and the second waveguide grating structure unit (now to be considered as a whole as a reference path), for example with a chemo-sensitive layer of different Specificity or with a non-specific chemo-sensitive layer with or without 'non-specific binding' (eg a dextran layer to which no recognition molecule is bound), for example, the size tF (measured in the signal path) - t can be used as the sensor signal _F (measured in the reference path).

Although the effective refractive indices N (TE) and N (TM) of the TE or TM mode may not be measured at the same location (however, the two sensing pads are covered with the same chemo-sensitive layer in the case of a chemo-sensitive sensor) with the waveguide grating structure unit with the interesting sensor signals S and / or ΔS

ΔS = Δα (TM) - Δα (TE) = Δ (α (TM) - α (TE)) (ΔS = Δ (α (TE) - α (TM))) (α = decoupling angle) ΔS = ΔN (TM) - ΔN (TE) (ΔS = ΔN (TE) - ΔN (TM)),

the layer thickness of the waveguiding film should preferably be chosen such that the sensitivity of the refractive index of the cover medium is ÖN (TE) / δnc = 5N (TM) / öric, which at least affects the influence of changes in the refractive index caused by temperature changes on the cover medium Sensor signal eliminated (the refractive index nc of the cover medium depends on the temperature, ie nc = nc (T), where T is the temperature),

ΔS = Δt _F = const ((δN (TE) / r3T) - ¹ ΔN (TE) - (f3N (TM) / aT) ^"1 ΔN (TM))

where const =

and the change in the layer thickness Δt _F from the system of equations

ΔN (TE) = (öN (TE) / cτtF) ΔtF + (r3N (TE) / f3T) ΔT (1)

ΔN (TM) = (r3N (TM) / r3t _F ) ΔtF + (δN (TM) / 5T) ΔT

is calculated and 5N / 5T is the temperature coefficient of the waveguide including cover (= sample) for the corresponding mode (SN / dT is determined experimentally (or according to theory) taking into account the temperature coefficient (dN / dT) _G RAτiNG of the grating (see also further below)),

ΔS = Δt _F , the layer thickness t _{F being} calculated using the three-layer waveguide model (with N (TE) and or N (TM) as an input parameter),

ΔS = Δt _A , the change in the additional layer thickness Δt _A from the equation system

ΔN (TE) = (dN (TE) / & _A ) Δt _A + (3N (TE) / f3T) ΔT (2)

ΔN (TM) = (f3N (TM) / Öt _A ) Δt _A + (dN (TM) / 5T) ΔT is calculated,

ΔS = Δt _A , the additional layer thickness t _{A being} calculated using the four-layer waveguide model (or five-layer waveguide model),

ΔS = Δr, the mass assignment T (see Chem. Commun. 1997, 1683-1684) with the four-layer waveguide model (or also with the three-layer waveguide model using approximations (see J. Opt. Soc. Am . B, Vol. 6, No.2, 209-220)) is calculated,

etc. are measured. The above sensor signals are interesting because they have a low temperature dependency. Basically, however, it must also be taken into account during the measurement that the grating period also exhibits a temperature dependency as a result of the thermal expansion coefficient of the sensor chip. ΔΛ = αΛΔT, where ΔΛ is the change in the grating period Gitter, ΔT is the change in temperature T and α is the coefficient of thermal expansion (typically 4.5 10 ^"6 K ^{" 1} for glass and 6.1 10 ^'5 K ^{' 1} for polycarbonate). That caused by the grid

= l (λ / Λ) α, where 1 is the diffraction order and λ is the wavelength, contributes to the temperature coefficient (dN / dT) c _H ip (= experimental measurement) of the complete sensor chip as follows: (dN / dT) cHπ> = (dN / dT) _A vEGuiDE +

+ (dN / dT) _G RAτiNG, where (dN / dT) _A vEGuiDE + SAMPLE is the temperature coefficient of the waveguide including the cover (= sample).

The sensor signals S and ΔS can be recorded as a function of time. But it is also possible to measure and compare only an initial and final state on a waveguide grating structure unit, for example, other waveguide grating structure units can be evaluated in between or the waveguide grating structure can even be removed from the measuring unit in between, since absolute angles or differences in angles (or distances) (Differences of distances) of light points) and these sizes with respect to a Tilt or shift remain stable. A measured value can be determined by a single measurement, but also by statistical evaluation (eg averaging) of several individual measurements.

If there are other disturbances besides temperature changes ΔT such as diffusion of sample liquid into the micropores of a porous waveguiding film (which leads to a change in refractive index Δn _{F of} the waveguiding film) or stresses (such as compressive stress or tensile stress) or stress changes or if at all only pore diffusion is present (in the first case under certain conditions (for example the temporal course of the pore diffusion with temperature T is known as a family of curve parameters)) (approximate) a (common) disturbance variable ξ with N (t _F , ξ) = N ( t _F , n _F (ξ), n _s (ξ), nc (ξ)) or N (t _A , ξ) = N (t _F , t _A , n _A (ξ), nF (ξ), n _s (ξ), nc (ξ)) are introduced. Equation (1) or (2) then takes the form

ΔN (TE) = (5N (TE) / r3S) ΔS + (öN (TE) / aξ) Δξ (3)

ΔN (TM) = (cN (TM) / dS) ΔS + (5N (TM) / öξ) Δξ

where ΔS is the sensor signal ΔtF (or Δt _A ) and, for example in the case of the three-layer optical fiber model oN / δξ = (r3N / f3n _F ) (δn _F / δξ) + (r3N / δns) (δns / r3ξ) + (aN / r3nc) (c δξ) (+ (cN / dt _F ) (dt _F / dζ) if t _F = t _F (ξ)). Logically follows directly from ξ = T formula (1) or formula (2). ξ = n _F describes, for example, diffusion at constant temperature.

Even if there are several faults, the three-layer waveguide model can be used and the mode equation can be solved (example: input: N (TE), N (TM), output: tF, n _F (= refractive index of the waveguiding film) or t _F , nc (= refractive index of the cover medium) or tF, n _s (= refractive index of the substrate)). In the three-layer waveguide model, waveguiding film, chemo-sensitive layer and specific binding partner are treated as one layer with a refractive index seeks. Although ΠF and nc (and possibly ns) are not exactly known and may change during the experiment due to the perturbation, the measured variable tF is relatively independent of the perturbation, but depends on the binding process. The disturbance is, so to speak, packed into the second output variable, the "refractive index" parameter.

Basically, pore diöusion and temperature fluctuations are independent phenomena and should be described by two disturbance variables ξi and ξ ₂ (generalization: k independent disturbances are described by k independent disturbance variables ξι, ..., ξk). From N (t _F , ξ _l .., ξk) it follows for both polarizations, for a given mode number and for a given wavelength

ΔN = (3N / & _F ) Δt _F + (cN / δξι) Δξ, + ... + (δN / aξ _k ) Δξ _t (4)

Since the disturbance variables can influence the waveguide parameters tF, ΠF, ns, nc, the following applies:

öN / δξi = (r3N / r3t _F ) ( _F / δξi) + (δN / n _F ) (δn _F / öξi) + (t5N / r3n _s ) (r3n _s / r3ξ _i ) + (aN / σ c) ( δnc / δξi)

(5)

(l <= i <= k) (The same applies to N (t _A , ξι, ..., ξk)) - For the pore diffusion (ξι = n _F ) and the temperature change (ξ ₂ = T) it follows (4)

ΔN = (dN / δt _F ) Δt _F + (SN / δnF) Δn _F + (dN / dT) ΔT (6)

for both polarizations, given mode number and given wavelength. δ / f5T can be determined experimentally, for example. For example, if both polarizations are measured, (6) represents two equations with three unknowns. However, if measurements are carried out at several wavelengths (taking into account the refractive index dispersions) and / or mode numbers, several systems of equations can be used. based on three equations and three unknowns. If the dispersion in Δnp (λ) is taken into account as an unknown (λ = wavelength), four equations with four unknowns Δt _F , Δn _F (λι), Δnp (λ ₂ ) are obtained, for example, with two wavelengths λi and λ ₂ and two polarizations. , ΔT and determines the unknowns from them. The size ΔtF forms the sensor signal.

Analogous to the above, the sensor signal t _F and the disturbance variables can also be determined from the (three-layer or four-layer) mode equations for the two polarizations and for several wavelengths and mode numbers, provided that the system of equations can be solved numerically.

In principle, the layer thicknesses to a certain extent also depend on the temperature (see also Applied Optics 20 (1981), 2280-2283), but this has been neglected in the approximation in formulas (1), (2) and (3). However, it is also possible to select such a combination of (several) layers and substrate with respect to at least one sample that the temperature coefficient of the waveguide or the grating coupler (or the waveguide grating structure) with respect to at least one sensor signal S (= α (TE ), α (TM), α (TM) - α (TE), N (TE), N (TM), N (TM) - N (TE), t _F , t _A etc.) is almost zero (Die However, sensitivity remains high). For example, one of the layers involved can be an SiO ₂ layer.

The absolute temperature coefficient with respect to a sensor signal S is dS / dT, the corresponding relative temperature coefficient is (l / S) (dS / dT). With dN (TE) / dT = 0 or dN (TM) / dT = 0, S = N (TE) or S = N (TM) also become interesting temperature-independent sensor signals.

If there is only one grid in a 'sensing pad', you can use the grid in the

European patent application 0 482 377 A2 described reflection arrangement

(possibly with a sensor chip with tilting of the grating level to the lower substrate level). The measurement with the reflection arrangement on weak or more modulated (monodiffractive or multidiffractive) waveguide gratings (homogeneous gratings, superimposed gratings with different grating periods and / or grating orientations, chirped gratings etc.) can be carried out for TE as well as for TM waves. The mode can be excited from the left, the reading from the right or vice versa. For example, the excitation of the TE wave can take place from the left and the excitation of the TM wave from the right in a sensing pad. In a second sensing pad (possibly with a different grating period and / or grating orientation), the excitation can take place in reverse to the first sensing pad. The reading can take place in the reflected and transmitted light field in zero or higher diffraction orders. It is thus also possible with the reflection arrangement to determine absolute coupling angles. The measured absolute coupling angle, which for the same grating period (and the same diffraction order) essentially corresponds to half the angular difference between the two corresponding resonances, does not change, even if the sensor chip is tilted slightly. From the absolute coupling angles for the TE wave or the TM wave, the corresponding effective refractive indices and other integrated optical measurement parameters such as the layer thickness t _{F of} the waveguiding film in the three-layer waveguide model etc. can be determined.

It is of course also possible that the excitation of the TE and TM waves from the left and the reading from the right (possibly only with a CCD) in the first 'sensing pad' and excitation in the second 'sensing pad' (possibly with a different grating period) and reading takes place in reverse.

It is also possible that the plane of incidence of the beam guide responsible for the reference path (second 'sensing pad') is rotated or tilted or rotated or tilted relative to the plane of incidence of the beam guide responsible for the signal path (first 'sensing pad'). The first and second 'sensing pad' can also coincide. The coupling angles for the TE wave and the TM wave can also be measured at different wavelengths and / or mode numbers. Another 'sensing pad' arrangement consists of two (identical) chirped grids (grids with a running grating period), one grating serving as the coupling grating and one grating serving as the coupling grating. Chirped gratings are known from the literature (see, for example, patent application WO 97/09594). The signal 'sensing pad' and the reference 'sensing pad' can have the same or opposite Chiφ direction (direction in which the grating period changes (eg increases) is perpendicular to the direction of the mode propagation). The 'sensing pads' can be covered with the same (bio) chemo-sensitive layer (see below) or have different ((bio) chemo-sensitive) layers, the ((bio) chemo-sensitive) layer of the second 'sensing pads' (reference 'sensing') pads') is another (bio) chemo-sensitive layer or a non-specific ((bio) chemo-sensitive) layer with or without 'nonspecific binding' (e.g. dextran without a recognition molecule) or a pure protective layer.

The chirped grating, which is responsible for the coupling, is illuminated with a (non-wedge-shaped or possibly (slightly) wedge-shaped) light band, of which a certain proportion, for which the coupling condition is fulfilled, is coupled into the waveguide. The coupling-chirped grating of signal 'sensing pad' and reference 'sensing pad' can also be used simultaneously (if necessary with a single longer light band) (if necessary with 45 ° linearly polarized or circularly (or elliptically) polarized light (for excitation of modes of both polarizations)) are illuminated. When modes of different polarization are excited at a fixed angle of incidence, the corresponding grating periods of the two sensing pads are different.

With the same polarization and opposite chirp direction of the signal 'sensing pad' and reference 'sensing pad' (both 'sensing pads' are coated with the same (bio) chemo-sensitive layer), the two out-coupled light points migrate due to the (bio) chemical bond (Practically) perpendicular to the direction of propagation of the modes towards or away from each other (depending on the chirp direction or chirp orientation). The position of the light points is with PSDs or (a) l- (or 2) dimensional CCD measured. The change in the effective refractive index of the corresponding polarization can be calculated by changing the distance between the two light points. The distance between the two light points is an absolute size, since the distance between the two light points is independent of shifts or small tilting. (If the signal and reference path have different polarization, the measurement signal ΔN (TM) - ΔN (TE) can be determined).

With the same polarization, the same Chiφ direction of the two 'sensing pads', but different (bio) chemo-sensitive layers (e.g. on signal path: specific layer, on reference path: non-specific layer), the distance between the two light points forms a referenced (absolute) sensor signal .

The arrangement of the penultimate paragraph can in turn be duplicated for the other polarization (by adapting the Gitteφeriode) and can in turn be understood as a reference path for the entire arrangement of the penultimate paragraph (now to be integrated as a signal path). The change in the effective refractive index of the other polarization can be measured here. (However, the polarization (s) remain the same and another chemo-sensitive layer (with or without nonspecific binding) or a non-specific layer with or without non-specific binding (e.g. dextran layer without recognition molecule) is used , the referenced measured variables (measured variable (first arrangement) - measured variable (second arrangement)) can be determined from the measured variables ΔN (TE) or ΔN (TM) or ΔN (TM) - ΔN (TE).

It is also possible that only the coupling-in grating is present as a chiφed grating, but the coupling-out grating is, for example, monodiffractive (or multi-diffractive). Due to the (bio) chemical bond, the two light spots from the penultimate paragraph no longer move perpendicular to the direction of the mode propagation, since the decoupling grating also deflects in the plane of incidence (perhaps better called the plane of the decay) (ie it changes the decoupling angle). If, in turn, work is carried out for signal 'sensing pad' and reference 'sensing pad' with three grids each (one coupling grating and two coupling grids or two coupling grids and one coupling grating), with modes being excited in the forward and reverse directions, is the coupling grating a chiφed grating (with the same or reversed Chiφ orientation between the two 'sensing pads') and the decoupling grids are, for example, monodiffiractive (or multidifiraactive), then shifts or tilting of the x and y axes (orientation of the axes as in Figure 1) identified and eliminated based on the absolute measurement. The same remarks apply to the chemo-sensitive layer (s) as to the 'sensing pad', which consists of two chiφed gratings. If, for example, only the (absolute) decoupling angle (and / or measurement variables derived from it) is considered as a sensor signal, the chiφed coupling grating can also be illuminated with a wedge-shaped incident light band. Here, too, the above arrangement (signal and reference path) can be duplicated and interpreted as a new reference path (with or without a different chemo-sensitive layer or with a non-specific layer) to the above arrangement (now to be understood collectively as a signal path).

The sample liquid (s) is (are) brought into contact with the waveguide or through a 'well' or a matrix of 'wells', a flow cell or a matrix of flow cells, a capillary cell or a matrix of capillary cells etc. brought the chemo-sensitive substance (s).

Two-dimensional arrangements lead, for example, to a 'microplate' with 96, 384, 1536 'wells' etc. (But other two-dimensional formats (eg disks) are also possible). The production of individual strips (one-dimensional arrangement) is also possible. The strips can also be used in the frame of a microplate, for example. The 'wells' can be applied as a separate sample cell (or sample cell plate) to the sensor chip plate which contains the waveguide grating structure units. However, it is also possible to provide the substrate itself with depressions in such a way that these depressions already function as the 'wells' or take over the flow cuvettes or the capillary cuvettes. In the latter two cases, the sensor chip plate must be covered with a cover plate provided with holes. In the first case, the sensor chip plate can be covered with a cover plate (without holes) in order, for. B. to avoid evaporation. The holes are used for supplying and removing the sample or for venting. Plastic substrates and injection molding techniques or hot embossing techniques are advantageously used. But sol-gel techniques, UV hardening techniques for organic / inorganic composites, glass embossing techniques (hot embossing or (injection) molding), etching techniques, laser ablation etc. also form an alternative.

The lattice structures can be made with embossing techniques (hot stamping, cold stamping, UV curing) or injection molding in plastic, sol-gel, glass, UN-curable organic or inorganic materials or organic / inorganic composites, ormocers or Νanomers, with laser ablation paired with interferometry, Holography and / or phase mask technology, photolithography paired with Νass or dry etching, with photopolymerization (see, for example, P. Coudray et al., Crit. Rev. Opt. Sei. Tech. (SPIE) CR68 (1997), 286- 303) or with casting techniques (e.g. in sol-gel) etc. Plastic injection molding techniques, such as those used in the production of compact disc discs (eg in polycarbonate), are particularly suitable. The lattice structure can be in (or on) the substrate or in (or on) a layer or in combinations thereof. The grating structures can be surface relief gratings (or interface relief gratings) or refractive index gratings (or volume gratings) or combinations thereof. The wave-guiding film can be a sol-gel layer (SiO ₂ -TiO ₂ , TiO ₂ , (non-porous) high-index lead silicate glass etc.), an organic / inorganic composite layer, a polymer layer, a PVD, a CVD, a PE-CND -, a PI-CVD layer, a photopolymerizable high-index layer (eg photopolymerizable TiO ₂ ) etc. or combinations thereof. It is also possible that a layer (preferably with a low refractive index, for example made of SiO ₂ sol gel, SiO ₂ -TiO ₂ sol gel (with a low TiO ₂ content), lead silicate glass sol gel (with a low lead content), float glass-like sol gel etc.) contains the lattice structure and a second layer (e.g. made of SiO ₂ -TiO ₂ , TiO, Ta ₂ Os, HfO ₂ , ZrO ₂ , Νb ₂ O ₅ , Si ₃ N ₄ , lead silicate glass etc.) forms the waveguiding film. Because the softening point of lead silicate glass is significantly below the softening point of glass, the lead silicate glass can be melted without damaging the glass. The melting process leads to almost pore-free lead silicate glass. If the substrate and the layer (s) have similar thermal expansion coefficients, the formation of microcracks can be prevented (microcracks increase the damping values of the modes). PVD and CVD processes allow the production of very compact waveguiding films.

The substrate can be made of plastic (e.g. polycarbonate, PMMA, polystyrene etc.), sol-gel or glass (float glass, slides, soda-lime glass, borosilicate glass, alkali-free glass, quartz etc.). However, the grid material can also be used as substrate material (e.g. Ormocer, UV curable material).

The sample cell can consist of depressions (holes, bores), but also of flow cells. These flow cells can also be configured such that the sample liquid can be supplied with the needle of a pipetting robot. It is also possible for a second sample cell consisting of flow-through cuvettes to be inserted into a first sample cell with depressions (holes).

Positioning marks (holes, depressions, pins, peaks, etc.) can be inserted or applied into the substrate or anywhere on the sensor chip plate. These positioning marks guarantee that the incident light beam hits the coupling grating (s). The positioning mark is identified via the measuring unit.

For the separation of the light wave carrying the sensor signal from other light waves (for example from the light wave reflected on the underside of the substrate), it is also advantageous if the wave-guiding film is not plane-parallel to the underside of the substrate. This non-plane parallelism can be in the form of wedges, prisms, cylindrical indices, spherical lenses, cylindrical lenses, etc. With the chemosensitive waveguide structure, not only direct detection but also marking detection can be carried out. The use of a 'refractive index label' (eg a plastic ball (eg latex ball), bio-chemical and biological fragments etc.) can already be seen as a proof of marking. It can be used to implement sandwich assays or competence assays, for example.

A monochromatic light source such as a (pulsed) laser, a (pulsed) laser diode, a (pulsed) LED with or without filter in the (infra) red, blue-green or ultraviolet spectral range is advantageously used as the light source. Thermal light sources with a filter can also be used. Red-blue-green or ultraviolet wavelengths have the advantage that (a) the sensitivity for direct detection increases and (b) in addition to 'direct sensing', fluorescence, phosphorescence and lurninescence tests can be carried out with the same light source, whereby the excitation the fluorescence, phosphorescence or luminescence occurs via the evanescent wave (in the form of a TE wave or TM wave or TE and TM wave). The fluorescent, phosphorescent and luminescent light can be observed as a plane or as a guided light wave. The fluorescence light (or its intensity) caused by the TE excitation wave can be referenced or compared with the fluorescence light (or its intensity) caused by the TM excitation wave, advantageously taking into account the intensity of the excitation wave and possibly the detectors by the Use of polarizers can be made sensitive to polarization. The guided fluorescence, phosphorescence and luminescence light wave can be coupled out via a grating and fed to a detector. Sandwich assays, competition assays etc. can be carried out, with at least one binding partner involved being fluorescence (phosphorescence, luminescence) -labeled. The (bio) chemosensitive layer can be on the grid, but can also only be present between the grids or outside the grid. Fluorescence, luminescence and phosphorescence measurements show a low temperature dependence. Purely inorganic waveguide grating structures are particularly suitable for fluorescence, luminescence and phosphorescence measurements (grating is made in glass or sol-gel (e.g. SiO ₂ ) or in inorganic waveguiding film, waveguiding film made of inorganic material (e.g. Si ₃ N or oxide layers such as TiO ₂ or Ta ₂ Os or lead silicate layers etc.). Inorganic materials, for example, show little inherent fluorescence. If you are working with a plastic substrate, it is advisable to apply an inorganic deep-refractive intermediate layer (eg SiO ₂ ) to the plastic substrate. The layer thickness of this intermediate layer is to be chosen so large that the evanescent wave running in it practically no longer "sees" the plastic substrate. As a result, at least the inherent fluorescence generated by the guided light wave is greatly reduced.

If the intensity of the (possibly decoupled) excitation wave as well as that of the (possibly decoupled) emission wave is measured, various interference factors (such as those caused by fluctuations in the intensity of the excitation wave) can be eliminated by referencing. The referenced sensor signal is then, for example, the intensity of the fluorescence (luminescence, phosphorescence) divided by the intensity of the excitation light wave. The intensity of the excitation wave can be measured before the excitation wave penetrates the chemo-sensitive layer or after the excitation wave emerges from the chemo-sensitive layer or at both points. With referenced markings, absolute kinetic measurements as well as absolute end point measurements can be carried out. In the case of absolute (end point) measurements, the (bio) chemical interaction on the waveguide grating structure can also take place outside the measuring instrument.

It should also be noted that with a (digital or analog) PSD (position sensitive detector) (1-dimensional or 2-dimensional), a photodiode array (PDA) (1-dimensional or 2-dimensional) or a CCD array (1 -dimensional or 2- dimensionally) not only (positions of) light field distributions but also intensities can be measured.

Direct detection as well as marking detection (on the basis of fluorescence, luminescence, phosphorescence) can (but need not) be carried out in part with the same detectors. Evanescence field excitation can also be combined with time-resolved measurement techniques (e.g. time resolved fluorescence or luminescence). In the case of time-resolved measurement techniques, the marking is stimulated with a pulsed light wave (predominantly in the visible or ultraviolet wavelength range). The decay times for the fluorescence (luminescence, phosphorescence) of free and bound marker molecules (with or without energy transfer between two different fluorescence molecules (see e.g. Förster theory in CIS Bio International, November 1995, Number 3)) are different. The depth of penetration of the guided excitation wave into the sample determines the "observation volume".

It is interesting that with the present waveguide grating structures, absolute measurements can be carried out both label-free and fluorescence (phosphorescence, luminescence) - labeled (if necessary also simultaneously). In both cases, there is no need for a moving mechanism for the light coupling. Of course, (continuous) kinetic measurements or real-time measurements can also be carried out on a non-absolute basis.

With a 'sensing pad' (signal path) (with or without a chemo-sensitive layer), light absorption measurements can also be carried out by measuring the intensities of the light beams (via grating, prism, taper or face) (possibly with the same detectors) even at different wavelengths become. The light absorption change can occur directly or indirectly (eg through enzymes) through the (bio) chemical interaction of the sample with the chemosensitive layer or through the sample itself or through the reactions taking place in the sample (with or without an additional reaction partner). The chemo-sensitive layer can be on the grid, between the grids or outside half of the grid. Fluctuations in the light intensity of the light source can be eliminated by referencing (eg with a beam splitter and a reference detector or via a non-coupled diffraction order and a reference detector). Referencing can also be done by covering the second 'sensing pad' with a protective layer and therefore not being able to interact with the sample. The referenced sensor signal is then the intensity of the signal path detector divided by the intensity of the reference path detector.

However, the protective layer can also be a (porous) (chemo-sensitive) layer which has no specificity (with or without 'non-specific binding') or specificity for another ligand.

Referencing can also (but does not have to) be done via half of a 'sensing pad' (e.g. signal path: mode in the forward direction, reference path: mode in the reverse direction).

Claims

PATENT CLAIMS

1. Optical sensor, comprising at least one optical waveguide with a substrate, waveguiding

Material, a cover medium and at least one waveguide grating structure, at least one light source by means of which light can be transmitted from the substrate side and / or from the cover medium side to at least part of the waveguide grating structure, and means for detecting at least two different light components, of which with at least one detection means Light emitted into the substrate and / or into the cover medium can be detected, with the waveguide being immovably fixable with respect to the at least one light source and the detection means in order to carry out a measurement.

2. Optical sensor according to claim 1, characterized in that the at least one waveguide grating structure contains at least two 'sensing pads', wherein a 'sensing pad' consists of at least one waveguide grating.

3. Optical sensor according to claim 2, characterized in that the two 'sensing pads' lie side by side.

4. Optical sensor according to claim 2 or 3, characterized in that the two 'sensing pads' differ in at least one Gitteφeriode.

5. Optical sensor according to one of claims 2-4, characterized in that the two 'sensing pads' lie one inside the other or one above the other.

6. Optical sensor according to claim 2 or 3, characterized in that the two 'sensing pads' have the same Gitteφeriode.

7. Optical sensor according to one of claims 2-6, characterized in that there is at least one grating in the volume and / or at an interface of a material contained in the waveguide.

8. Optical sensor according to one of claims 2-7, characterized in that a 'sensing pad' of the waveguide grating structure unit contains two coupling grids and a coupling grating arranged between the coupling grids, or two coupling grids and a coupling grating arranged between the coupling grids.

9. Optical sensor according to one of claims 2-8, characterized in that a 'sensing pad' of the Wellerdeit grating structure unit contains at least one chiφed grating.

10. Optical sensor according to one of claims 2-9, characterized in that at least one 'sensing pad' is at least partially covered with a chemo-sensitive or biochemo-sensitive layer.

11. Optical sensor according to claim 10, characterized in that at least a first 'sensing pads' and a second 'sensing pad' are each covered with a chemo-sensitive or biochemo-sensitive layer, the chemo-sensitive or biochemo-sensitive layers assigned to the 'sensing pads' having different specificities exhibit.

12. Optical sensor according to claim 10, characterized in that at least a first 'sensing pad' and a second 'sensing pad' are covered with a chemo-sensitive or biochemo-sensitive layer, the chemo-sensitive or biochemo-sensitive layer assigned to the first 'sensing pad' being specific for shows a ligand with or without 'non-specific binding', while the chemosensitive or biochemically sensitive layer has no specificity at all with or without 'non-specific binding'.

13. Optical sensor according to one of claims 1-12, characterized in that a 'well' or a matrix of 'wells' is applied to the waveguide grating structure or is introduced into the waveguide grating structure.

14. Optical sensor according to one of claims 1-13, characterized in that a flow cell or a matrix of flow cells or a capillary cell or a matrix of capillary cells is applied to the waveguide grating structure or is introduced into the waveguide grating structure.

15. Optical sensor according to one of claims 1-14, characterized in that the substrate and the materials contained in the waveguide have similar thermal expansion coefficients to avoid microcracks.

16. Optical method for characterizing and / or detecting a chemical and / or biochemical substance in a sample by means of an optical waveguide containing at least one waveguide grating structure, the sample being brought into contact with the waveguide in at least one contact zone, in the waveguide in the region of the at least one contact zone, at least one light wave is excited via the waveguide grating structure, which causes at least one light wave to interact with the sample, light in at least two different proportions, at least a portion of which comes from the area of the at least one contact zone, and at least one absolute one Measurement signal is generated by evaluating the detected light.

17. The method according to claim 16, characterized in that at least two light waves are excited in the waveguide in the region of the at least one contact zone which differ in polarization, in the number of modes and / or in the wavelength.

18. The method according to claim 16 or 17, characterized in that the measurement signal is generated on the basis of direct detection.

19. The method according to claim 18, characterized in that the measurement signal is a layer thickness or a layer thickness change according to the solution of the mode equations for at least one polarization, at least one wavelength and at least one mode number.

20. The method according to claim 18, characterized in that the measurement signal is a layer thickness or a layer thickness change according to the solution of a linear system of equations for at least one polarization, at least one wavelength and at least one mode number.

21. The method according to claim 16 or 17, characterized in that the measurement signal is generated on the basis of a marking verification.

22. The method according to claims 18 and 20, characterized in that both a measurement signal belonging to the direct detection and a measurement signal belonging to the marking detection are generated.

23. The method according to any one of claims 16-22, characterized in that the measurement signal is generated in a time-resolved manner.

24. The method according to any one of claims 16-23, characterized in that a waveguide or a waveguide grating structure is selected such that the temperature coefficient of the waveguide or the waveguide grating structure is almost zero with respect to at least one sample and with respect to at least one measurement signal.